The overall goal of our continuing research program is to develop advanced technologies based on electrospray ionization (ESI) with mass spectrometry (MS) to support structural biology efforts. The role of protein assemblies in normal cellular processes and especially diseases warrants a practical and sensitive method for their study and their structural elucidation. The superior sensitivity of mass spectrometry and the special ability of ESI to transfer solution-phase molecules to their gas-phase ionized counterparts without disrupting covalent bonds and maintaining weak non-covalent interactions are key for the study of protein complexes. ESI-MS can measure proteins and complexes in aqueous solution at near neutral pH, i.e., native MS. Moreover, top-down MS, the direct fragmentation of intact gas-phase large molecules to generate structure-informative product ions, has value for the structural characterization of protein complexes. Advanced techniques will be developed to facilitate the characterization of non-covalent protein assemblies. Novel methodologies and tools will be developed to improve the applicability of native MS. A critical barrier to membrane protein characterization by MS will be addressed. Methods to improve the efficiency and to expand the applicability of top-down MS for determining the binding sites of small molecule ligands on protein targets, and for elucidating the binding interface between large macromolecules will be developed. Enhancing multiple charging generated by ESI increases the efficiency for top-down MS and native MS. Combining top-down MS and hydrogen-deuterium exchange can deliver important structural information for protein complexes, such a protein-DNA/RNA complexes. The ability to enhance ESI charging for a variety of analytical platforms for MS will increase the applicability of ESI-MS. Top-down MS of protein-ligand and protein-protein complexes should deliver information on ligand binding location and protein-protein interfaces in a rapid manner that can be useful for biological systems of importance in biology, medicine, and human health. In the development of novel inhibitors, localization of their sites of interaction on the target of interest is a critical inital step in the drug discovery process. Our work to develop new methods for directly determining ligand binds sites may lead to a new strategy for screening potential drugs. Improvements in MS-based technologies can advance our views of how proteins and protein machines drive biology. Native MS will deliver important data that can be integrated with data derived from other biophysical techniques to generate 3D structural models to address relevant biological questions.